Process for producing hydrazinomonosaccharide derivatives and use thereof

ABSTRACT

A process for producing hydrazinomonosaccharide derivatives and use of hydrazines in determining the structures of aldose and ketose monosaccharides located at the reducing ends of saccharides.

TECHNICAL FIELD

[0001] The present invention relates to a method for producing hydrazinomonosaccharide derivatives which is useful for analyses ofmonosaccharides and use thereof. Specifically, the present inventionrelates to use of hydrazines for the production of hydrazinomonosaccharide derivatives and for the determination of structures ofaldose or ketose monosaccharides at reducing ends of saccharides.

BACKGROUND ART

[0002] Saccharides (also called sugars or carbohydrates) are maincomponents of biological systems. Saccharides constitute about 80% ofdry weight of plants and, either as monomers (monosaccharides) orpolymers consisting of monosaccharides covalently bound each other(oligosaccharides), are indispensable components of metabolic pathwaysin higher animals. In addition, saccharides are often found as parts oflarger biological macromolecules (including proteins, lipids and nucleicacids). Saccharides in such various forms have numbers of importantfunctions in nature.

[0003] A means of identifying a free monosaccharide or a monosaccharideas a monomeric component of an oligosaccharide is very useful because ofthe importance of saccharides in biological systems. Furthermore, ameans of identifying a monosaccharide at a reducing end of anoligosaccharide is important for the structural analysis of theoligosaccharide.

[0004] Recently, a method for analyzing a structure of anoligosaccharide was disclosed in WO 96/17824 (JP-A 11-501901). In themethod, a monosaccharide at a reducing end of an oligosaccharide isconverted to an N,N′-diacetylhydrazino monosaccharide derivative, andthe derivative is identified by gas chromatography/mass spectrometry(GC/MS) or the like. Theoretically, the types of monosaccharides at thereducing ends of all oligosaccharides can be identified according tothis method.

[0005] However, there is a problem that one can use the above-mentionedmethod only for an isolated saccharide. If this method is applied to amixture of several kinds of saccharides, it is impossible to determinethe saccharides from which the respective resulting several kinds ofhydrazino monosaccharides derive. Thus, if a naturally occurringsaccharide is to be analyzed according to this method, it is necessaryto isolate the saccharide beforehand.

[0006] For example, since a protein or a nucleic acid itself hasultraviolet absorbance, it is possible to trace the position duringseparation/purification procedures based on the absorbance. However,since a saccharide does not have such absorbance, it is desired to labelthe saccharide in order to facilitate separation/purification. Forexample, labeling with a fluorescent dye is suitable for this purpose.If a saccharide coexists with other contaminants derived from a naturalsource (e.g., proteins, nucleic acids, etc.), the saccharide having alabel should be readily distinguished from other contaminants. Thus, inthis case, a saccharide must be selectively labeled such thatcontaminating components other than the saccharide are not labeled.

[0007] A method for labeling a reducing residue of a saccharideexemplifies a method for selectively labeling a saccharide withoutlabeling other components derived from a natural source. Examples ofreducing residues include a carbonyl group at a reducing end of asaccharide and a free aldehyde group. Reactions on reducing residues ofsaccharides include a reductive amination reaction and a hydrazidationreaction. These reactions are irreversible. A method in which2-aminopyridine is used (S. Hase, T. Ibuki and T. Ikenaka, Journal ofBiochemistry, 95, 197-203 (1984)) exemplifies a method for labeling asaccharide using a reductive amination reaction. A method in whichBiotin-x-hydrazide (Calbiochem) is used (B. Ridley, D. Mohnen et al.,Analytical Biochemistry, 249, 10-19 (1997)) exemplifies a method forlabeling a saccharide using a hydrazidation reaction. In the lattermethod, a saccharide labeled with biotin through a hydrazide bond isprepared by forming a hydrazone by a reaction of a saccharide having areducing end with Biotin-x-hydrazide and then conducting a reductionreaction.

[0008] A free reducing end of a saccharide is utilized in the methoddisclosed in WO 96/17824. Therefore, it is impossible to directly use amonosaccharide isolated by labeling a reducing end of a saccharideaccording to this method for identifying the type thereof.

[0009] Furthermore, determination of the position of binding to aneighboring monosaccharide (hereinafter also referred to as the“substitution position”) is desired in addition to identification of thetype of a monosaccharide in order to precisely determine the structureof a monosaccharide at a reducing end of a saccharide. In other words,it is desired to determine the position of a hydroxyl group of aneighboring monosaccharide through which a monosaccharide at a reducingend binds. Only a technique for identifying the type of a monosaccharideat a reducing end of a saccharide is disclosed in WO 96/17824. Atechnique for determining the substitution position is not mentionedtherein.

[0010] A methylation analysis is known as a method for determining theposition of binding between monosaccharides constituting a saccharide. Ageneral methylation analysis comprises methylation of anoligosaccharide, hydrolysis of the methylated oligosaccharide, reductionof a free methylated monosaccharide, acetylation of a methylatedalditol, and an analysis of a partially methylated alditol acetate(PMAA) in this order. Fragmentation patterns for partially methylatedalditol acetates upon mass spectrometric analyses and rules thereof havebeen studied in detail for a long time, and it is possible to identifythe position of an acetyl group based on the fragmentation pattern (B.Lindberg, Methods in Enzymology, Vol. 28, pp. 178-195 (1972)). PMAAs aregenerated for all monosaccharides constituting an oligosaccharideaccording to a general methylation analysis. They are usually analyzedusing gas chromatography/mass spectrometry (GC/MS) equipment. Theposition of an acetyl group can be identified based on the fragmentationpattern upon a mass spectrometric analysis. On the other hand, one hasto rely on identification by comparison with a standard substance on gaschromatography for identification of the type of the monosaccharide. Forthis purpose, it is required that all possible PMAAs for allmonosaccharides constituting an oligosaccharide have been provided asstandard substances. It requires a lot of labor to prepare possiblePMAAs for all naturally occurring monosaccharides. In addition, it ispractically impossible to conduct chromatography that can be used toseparate and identify all possible PMAAs for all naturally occurringmonosaccharides.

OBJECTS OF INVENTION

[0011] The main object of the present invention is to provide a meansthat enables determination of the type and the substitution position ofa monosaccharide at a reducing end of a saccharide even if thesaccharide is not isolated.

SUMMARY OF INVENTION

[0012] The present inventors have studied intensively in order toachieve the above-mentioned object. As a result, the present inventorshave found that it is possible to determine the type of a monosaccharideat a reducing end of a saccharide according to a method similar to themethod as described in WO 96/17824 by converting the saccharide into ahydrazino monosaccharide derivative even if the saccharide is notisolated. Thus, the present invention has been completed.

[0013] Accordingly, the present invention provides the following:

[0014] (1) a method for producing a hydrazino monosaccharide derivative,the method comprising at least:

[0015] (a) reacting a saccharide having a reducing end with a hydrazineof formula (I) to produce a hydrazone:

NH₂—NR¹(R²)   (I)

[0016] wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

[0017] (b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative; and

[0018] (c) cleaving the hydrazino derivative obtained in step (b) by thereaction that can cleave a glycosidic linkage to obtain a hydrazinomonosaccharide derivative;

[0019] (2) the method according to (1), which comprises N-acetylatingthe hydrazino derivative obtained in step (b) before subjecting it tostep (c);

[0020] (3) the method according to (1) or (2), which comprisesmethylating a hydroxyl group of the hydrazino derivative obtained instep (b) or an N-acetylation product of the hydrazino derivative beforesubjecting it to step (c);

[0021] (4) the method according to any one of (1) to (3), wherein R¹ isan acyl group;

[0022] (5) the method according to any one of (1) to (4), wherein R¹ isan acyl group that has a ultraviolet or visible-absorbing substance, afluorescent dye or an immobilization support as its portion;

[0023] (6) a method for identifying a monosaccharide at a reducing endof a saccharide having a reducing end and/or for determining a positionof binding of a monosaccharide at a reducing end to a neighboringmonosaccharide, the method comprising at least:

[0024] (a) reacting a saccharide having a reducing end with a hydrazineof formula (I) to produce a hydrazone:

NH₂—NR¹(R²)   (I)

[0025] wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

[0026] (b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative;

[0027] (c) cleaving the hydrazino derivative obtained in step (b) by thereaction that can cleave a glycosidic linkage to obtain a hydrazinomonosaccharide derivative;

[0028] (d) completely acetylating the hydrazino monosaccharidederivative obtained in step (c); and

[0029] (e) identifying the completely acetylated hydrazinomonosaccharide derivative obtained in step (d);

[0030] (7) the method according to (6), which comprises N-acetylatingthe hydrazino derivative obtained in step (b) before subjecting it tostep (c);

[0031] (8) the method according to (6) or (7), which comprisesmethylating a hydroxyl group of the hydrazino derivative obtained instep (b) or an N-acetylation product of the hydrazino derivative beforesubjecting it to step (c), wherein a position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide isdetermined in step (e);

[0032] (9) the method according to any one of (6) to (8), wherein theidentification in step (e) is carried out using gas chromatography/massspectrometry (GC/MS);

[0033] (10) a method for labeling a saccharide having a reducing end,the method comprising at least:

[0034] (a) reacting a saccharide having a reducing end with a hydrazineof formula (I) to produce a hydrazone:

NH₂—NR¹(R²)   (I)

[0035] wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a chemical reaction that can cleavea glycosidic linkage; and R² is hydrogen or an alkyl group containing1-8 carbon atoms;

[0036] (b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative; and

[0037] (c) N-acetylating the hydrazino derivative obtained in step (b);

[0038] (11) a hydrazine of formula (I) used in the method defined by anyone of (1) to (10):

NH₂—NR¹(R²)   (I)

[0039] wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms;

[0040] (12) a kit for producing a hydrazino monosaccharide derivative,which contains the hydrazine defined by (11);

[0041] (13) a kit for identifying a monosaccharide at a reducing end ofa saccharide and/or for determining a position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide, whichcontains the hydrazine defined by (11);

[0042] (14) a kit for labeling a saccharide having a reducing end, whichcontains the hydrazine defined by (11);

[0043] (15) a saccharide of formula (III):

R⁴—N(Ac)—NHR¹   (III)

[0044] wherein Ac is an acetyl group; R¹ is a group other than hydrogenthat has a detectable label and/or an immobilization support as itsportion or can bind to a detectable label and/or an immobilizationsupport; the bond between R¹ and N is a bond that is cleavable by achemical reaction that can cleave a glycosidic linkage; R⁴ is a groupthat may have a glycosidic linkage with a saccharide and from which onehydrogen atom linked to the C-1 position of a 1-deoxy aldose or onehydrogen atom linked to the C-2 carbon of a 2-deoxy ketose is removed,excluding a case where R¹ is an acetyl group, and R⁴ is a group thatdoes not have a glycosidic linkage with a saccharide and from which onehydrogen atom linked to the C-1 position of a 1deoxy aldose or onehydrogen atom linked to the C-2 carbon of a 2-deoxy ketose is removed;and

[0045] (16) the saccharide according (15), wherein R¹ is an acyl group.

[0046] According to the present invention, it is possible to identifythe type of a monosaccharide at a reducing end of a saccharide even ifthe saccharide is not isolated. Furthermore, a position of binding to aneighboring monosaccharide can be determined by methylating a hydroxylgroup of a hydrazino derivative as in the aspect of (7). Theidentification or determination as described above can be carried outwithout a need of separation of a reaction product from a reactionmixture by using a hydrazine that has an immobilization support as itsportion or contains a group that can bind to an immobilization support.

BRIEF DESCRIPTION OF DRAWINGS

[0047]FIG. 1 illustrates thin-layer chromatography on whichbenzoylhydrazine derivatives of N-acetyl lactosamine which had beenallowed to stand under various conditions and N-acetylation productsthereof were developed.

DETAILED DESCRIPTION OF THE INVENTION

[0048] A hydrazino monosaccharide derivative produced according to themethod of the present invention is used in a method for identifying amonosaccharide at a reducing end as described in WO 96/17824 (JP-A11-501901, incorporated herein by reference). According to the method asdescribed in WO 96/17824, a hydrazone is produced by reacting anisolated saccharide having a reducing end with a hydrazine, thehydrazone is reduced to a hydrazino derivative, a hydrazinomonosaccharide derivative is optionally cleaved from an oligosaccharide,the hydrazino monosaccharide derivative is acetylated to obtain anN,N′-diacetylhydrazino monosaccharide derivative, and thediacetylhydrazino monosaccharide derivative is identified by means ofGC/MS or the like.

[0049] There is no specific limitation concerning the saccharide havinga reducing end used in step (a) of the method for producing a hydrazinomonosaccharide derivative of the present invention. It may be anysaccharide of which the monosaccharide at the reducing end is to beidentified and/or the position of binding of the monosaccharide at thereducing end to a neighboring monosaccharide is to be determined. Suchsaccharides include monosaccharides, oligosaccharides andpolysaccharides as well as mixtures thereof. A sample containing asaccharide used in the method of the present invention may furthercontain other components derived from a natural source (proteins,nucleic acids, etc.). A method for obtaining a sample containing asaccharide used in the method of the present invention from a naturalsource is described, for example, in “Seikagaku jikken koza 4—Toshitsuno kagaku (Jo)”, edited by the Japan Biochemical Society, published onApr. 12, 1976, Tokyo Kagaku Dozin.

[0050] As used herein, the phrase “a monosaccharide at a reducing end”refers to a monosaccharide having a reducing property located at aterminus (a reducing end) of a saccharide in which the C-1 position ofan aldose or the C-2 position of a ketose is not subjected tosubstitution. An “aldose” refers to either a free monosaccharide or amonosaccharide at a reducing end of an oligosaccharide that may have analdehyde group at the C-1 position. A “ketose” refers to either a freemonosaccharide or a monosaccharide at a reducing end of anoligosaccharide that may have a ketone group at any one of internalcarbon atoms along the backbone of the monosaccharide.

[0051] A hydrazine that is structurally different from the hydrazineused in the method as described in WO 96/17824 is used in the method forproducing a hydrazino monosaccharide derivative of the presentinvention. Thereby, the need of isolating a saccharide to be analyzedbeforehand is eliminated. A “hydrazine” generally means a compoundgenerated by substituting an organic group for a hydrogen atom ofhydrazine (in a narrow sense, N₂H₄). A “hydrazine” used in the methodfor producing a hydrazino monosaccharide derivative of the presentinvention is any known or novel compound represented by formula (I):

NH₂—NR¹(R²)   (I)

[0052] wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms. Only one of four hydrogen atoms of hydrazine (in a narrowsense, N₂H₄) in the hydrazine used in the method as described in WO96/17824 may be replaced by an alkyl group containing 1-8 carbon atoms.

[0053] A “detectable label” is any label known in the art thatfacilitates the purification of a hydrazino derivative. Examples thereofinclude ultraviolet or visible-absorbing labels (e.g., benzene andderivatives thereof), fluorescent labels (e.g., fluoresceine, pyrene,anthracene and derivatives thereof), and radioactive labels (e.g.,radioactive hydrogen, radioactive carbon and radioactive iodine) as wellas a biotin label and a digoxigenin label. If it is desired that adetectable label would be attached after a hydrazino derivative isproduced, a hydrazine containing a group that can bind to a detectablelabel and a detectable group may exist independently. Alternatively, agroup that has a detectable label as its portion may be used. An acylgroup that has a fluorescent dye as its portion exemplifies a group thathas a detectable label as its portion. For example, a hydrazinoderivative can be readily purified by using a method known in the artsuch as normal phase high performance liquid chromatography usingfluorescence emitted from pyrene attached using 1-pyrenebutanoic acid,hydrazide (commercially available from Molecular Probes) as an index. Inaddition, many compounds such as biotin-hydrazide (Dojindo),benzoylhydrazine (Tokyo Kasei Kogyo) Cascade Blue hydrazide (MolecularProbes) are available as acylhydrazides having detectable groups.

[0054] Those skilled in the art can readily obtain a hydrazine compoundto be used for labeling a detectable label by synthesizing it. Forexample, an acylhydrazide can be readily obtained by attaching acompound having carboxylate as a functional group to hydrazine (in anarrow sense, N₂H₄) using a technique used for peptide synthesis(Izumiya et al., “Peptide gousei no kiso to jikken” (1985) Maruzen). Acompound having an amino group as a functional group can be condensedwith hydrazine (in a narrow sense, N₂H₄) according to theabove-mentioned method, for example, after the functional group isconverted to carboxylate using succinic anhydride. In addition,hydrazine (in a narrow sense, N₂H₄) can be introduced to a compoundhaving a hydroxyl group as a functional group by halogenation followedby a reaction with carbohydrazide (H₂N—NH—CO—NH—NH₂).

[0055] The above-mentioned techniques for synthesizing a hydrazinecompound can be applied not only in case of a detectable labelingcompound but also in case of an immobilization support.

[0056] Any or all of reaction steps of the method of the presentinvention may be conducted in a liquid phase or in a solid phase. An“immobilization support” may be used for conducting various reactions onhydrazino derivatives in a solid phase. For example, a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide can be determined in step (e) of the method foridentifying a monosaccharide at a reducing end and/or for determining aposition of binding of a monosaccharide at a reducing end to aneighboring monosaccharide of the present invention as described belowin detail. The determination can be accomplished by methylating ahydroxyl group of the hydrazino derivative obtained in step (b) beforesubjecting the hydrazino derivative to step (c). In general, themethylation reaction involves several steps of reaction/washing. Aprocedure for separating a reaction product from a reaction mixture ineach step can be omitted by attaching a hydrazino derivative to animmobilization support.

[0057] If a hydrazino derivative attached to an immobilization supportcan be purified, the saccharide to be used need not be isolated.Generally, a hydrazine that has an immobilization support as its portionor contains a group capable of binding to an immobilization support isused for an isolated saccharide. If a saccharide is not isolated, onecan label a mixture containing the saccharide with a hydrazine compoundcontaining a detectable label, purify a hydrazino-derivatized saccharideusing the label as an index, and attach the purifiedhydrazine-derivatized saccharide to an immobilization support. Forexample, if a saccharide is reacted with an excess amount of4-aminobenzhydrazide, the saccharide is preferentially condensed with ahydrazide group to form a hydrazone. After reduction, a saccharidehydrazino derivative can be purified using ultraviolet absorbance of abenzene ring as an index. The purified saccharide hydrazino derivativecan be attached through an amino group of the derivative, for example,to an immobilization support having N-hydroxy succinimide ester as afunctional group. Immobilization supports include glass beads, polymermatrixes, sintered glass disks, fiber glass membranes and polymermembranes. It is usually desirable that the immobilization support has afunctional group for immobilizing a hydrazine or a saccharide hydrazinoderivative. Examples of such functional groups include an amino group, acarboxyl group, a hydroxyl group and an alkyl halide group. Examples ofimmobilization supports having such functional groups include NovaSyn TGbromo Resin commercially available from Nova Biochem and Bio-Rex 70Resin commercially available from Bio-Rad. A hydrazine having such animmobilization support as its portion is produced, for example,according to the procedure as described in Example 2. The reducing powerof the thus obtained hydrazine can be measured according to a methodknown in the art such as the Park-Johnson method (Park, J. T. andJohnson, M. J., J. Biol. Chem., 181, 149-151 (1949)).

[0058] The reaction of a saccharide having a reducing end with ahydrazine in step (a) is conducted under appropriate conditions known tothose skilled in the art, for example, in an appropriate solvent (e.g.,DMSO or acetonitrile) at 40-90° C. for 0.1-20 hours. For example, thereaction is conducted by heating in dimethyl sulfoxide (DMSO) containing10% acetic acid at 90° C. for 1 hour.

[0059] The reduction of a hydrazone to a hydrazino derivative in step(b) can be conducted using any appropriate reducing agent known to thoseskilled in the art. Examples of appropriate reducing agents includeboron hydride reagents, boron-centered hydrides, borane/diborane,aluminum hydride reagents, and other aluminum-centered hydrides havingalkoxy groups that cause substitution with covalently bound carbon orhydrogen. Catalytic hydrogenation can be conducted using hydrogen gasand one of various metals or a prepared alloy such as Raney nickel (anickel-aluminum alloy). In addition, dissolution of a reduced metal, useof an alkaline metal (lithium, sodium or potassium) and, for example,zinc, magnesium, tin, iron or mercury in a solvent (e.g., an alcohol,acetic acid, liquid ammonia or an ether such as 1,2-dimethoxyethane) aregenerally effective.

[0060] A reduction reaction is conducted in an appropriate solvent(e.g., DMSO or water) at 20 to 90° C. for 1 to 20 hours. For example, itis conducted by heating in a solution containing 2.5 Mborane-dimethylamine complex and 30% acetic acid in DMSO at 80° C. for 1hour or by allowing to stand in a 1 M sodium boron hydride aqueoussolution at room temperature for 16 hours. Those skilled in the artunderstand that the time required for such a reaction may be shortenedor prolonged depending on the elevation or lowering of temperature. Theproduct and the yield in a reduction step can be monitored using ananalytical technique such as proton NMR or mass spectrometry.

[0061] It is well known to those skilled in the art that steps (a) and(b) may be carried out not only as separate steps but also as stepsproceeding in parallel.

[0062] Furthermore, a hydrazino derivative may be N-acetylated after areduction reaction, if necessary. It is expected that the N-acetylationof a hydrazine derivative is effective in loss of charge and chemicalstabilization.

[0063] A hydrazino derivative is cleaved by a reaction that can cleave aglycosidic linkage to obtain a hydrazino monosaccharide derivative instep (c). The bond between R¹ and N contained in the hydrazine used instep (a) is also cleaved at the same time. Any reaction that can cleavea glycosidic linkage known to those skilled in the art can be used (see,for example, Biermann, C. J., Advances in Carbohydrate Chemistry andBiochemistry, Vol.46, 251-271). R¹ is appropriately selected by thoseskilled in the art such that the bond between R¹ and N can be cleavedunder the selected reaction conditions. In one embodiment, R¹ is an acylgroup. In another embodiment, R¹ is an acyl group that has a detectablelabel (a fluorescent dye or an immobilization support) as its portion.The step of cleavage can be accomplished by using acidic conditions inone of various solvents such as water, an alcohol or carboxylic acid.Appropriate cleaving agents include a solution of hydrochloric acid ortrifluoroacetic acid in water, hydrochloric acid in absolute methanol,and sulfuric acid in an acetic anhydride solution. The cleavage isgenerally conducted at 50 to 110° C. for 1 to 10 hours. For example, thecleavage is conducted by heating in 5% hydrochloric acid-methanol at 90°C. for 4 hours or by heating in 4 M hydrochloric acid at 100° C. for 4hours.

[0064] A hydrazino monosaccharide derivative obtained by a methodcomprising steps (a) to (c) is represented by formula (II):

R³—NH—NH—R²   (II).

[0065] Therein, R³ is a 1-deoxy aldose moiety or a deoxy ketose moietycovalently bound to N in the formula. If R³ is a 1-deoxy aldose moiety,the covalent binding to N is generated through the C-1 position. If R³is a deoxy ketose moiety, the covalent binding to N is generated throughthe deoxy carbon in the sugar backbone (which carbon is originallypresent in the ketone group). In all cases, R² is hydrogen or an alkylgroup containing 1-8 carbon atoms.

[0066] Among hydrazino monosaccharide derivatives of formula (II), ahydrazino monosaccharide derivative having a structure in which all orsome of hydroxyl groups of the 1-deoxy aldose moiety or the deoxy ketosemoiety represented by R³ are methylated is designated as an“O-methylated hydrazino monosaccharide derivative” in particular.

[0067] The aldose or the ketose constituting the group represented by R³may be any monosaccharide in a free form or at a reducing end of anoligosaccharide. Examples of aldoses include the following: aldohexoseseach containing 6 carbon atoms (e.g., D-glucose, L-glucose, D-allose,D-altrose, D-galactose, D-gulose, D-idose, D-mannose and D-talose);aldopentoses each containing 5 carbon atoms (e.g., D-arabinose,D-lyxose, D-ribose and D-xylose); aldotetroses each containing 4 carbonatoms (e.g., D-erythrose and D-threose); and aldotrioses each containing3 carbon atoms (e.g., D-glyceraldehyde). Ketoses include ketohexoseseach containing 6 carbon atoms (e.g., D-fructose, D-psicose, D-sorboseand D-tagatose).

[0068] Using the hydrazino monosaccharide derivative obtained asdescribed above, identification of a monosaccharide at a reducing end ofa saccharide having a reducing end and/or determination of a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide is carried out as follows.

[0069] A hydrazino monosaccharide derivative is completely acetylated instep (d). The term “acetylation” refers to covalent binding of one ormore acetyl groups to a molecule. A “completely acetylated” molecule isa molecule in which all of the free hydroxyl groups and the nitrogenatoms are acetylated.

[0070] Any appropriate O-acetylation reaction known in the art can beused for completely acetylating the derivative. Such reactions include,but are not limited to, reactions with an acetic anhydride/pyridinemixture, acetic anhydride, zinc chloride, sodium acetate, sulfuric acid,or acetyl chloride in a pyridine solution (see, for example, Horton D.IA, “The Amino Sugars”, pp. 3-211, R. W. Jeanloz (ed.), Academic Press,1969; incorporated herein by reference).

[0071] N-acetylation takes place more readily than O-acetylation.Therefore, complete acetylation of deoxyhydrazino alditol anddeoxy-(N′-alkyl hydrazino) alditol takes place under the conditions asdescribed above for “O-acetylation” to generate acetyl groups on all ofthe nitrogen atoms and the free hydroxyl groups in the molecule.

[0072] For example, O-acetylation is carried out by adding a 2:1 mixtureof pyridine and acetic anhydride to a sample and incubating theresulting mixture at 37° C. for 16 hours.

[0073] If N-acetylation is to be conducted selectively without effectingO-acetylation, the N-acetylation is conducted, for example, by mixingwith acetyl anhydride in a weak alkaline buffer. Saturated sodiumbicarbonate is preferably used as a weak alkaline buffer. Unlessotherwise stated, as used herein, “N-acetylation” means acetylationselective for nitrogen atoms.

[0074] The hydrazino derivative obtained in step (b) may be methylatedat the hydroxyl group before subjecting it to step (c) in the method ofthe present invention. As a result of the methylation, it is possible todetermine the position of binding of a monosaccharide at a reducing endto a neighboring monosaccharide (or the “substitution position”) in step(e).

[0075] The “methylation analysis” by which the position of bindingbetween monosaccharides constituting a saccharide is determined isuseful for determining a position of binding of a monosaccharide at areducing end to a neighboring monosaccharide. According to this method,a free hydroxyl group of a reducing sugar is first methylatedcompletely, the resulting methylated saccharide is cleaved to generate apartially methylated monosaccharide in which only the hydroxyl group atthe position of binding to a neighboring monosaccharide is free, thepartially methylated monosaccharide is acetylated, and the position ofacetylation is identified. Methylation is conducted by any method knownin the art. For example, methylation can be conducted using the methodof Hakomori (S. Hakomori, J. Biochem. (Tokyo), Vol.55, 205-208 (1964)),the DMSO—NaOH method (I. Ciucanu and F. Kerek, Carbohydrate Research,Vol.131, 209-217 (1984)), or the method of Anumula et al. (an improvedDMSO—NaOH method) (Anumula, K. R. and Taylor, P. B., Anal. Biochem.,Vol.203, 101-108 (1992)) (incorporated herein by reference).

[0076] According to the method for determining a position of binding ofa saccharide at a reducing end to a neighboring monosaccharide of thepresent invention, if R₁ is an acyl group and the DMSO—NaOH method orthe method of Anumula et al. is used as a means of methylation, thecompletely acetylated hydrazino monosaccharide derivative cannot beidentified later in step (e) unless the nitrogen atom (N) directly boundto the sugar of the hydrazino derivative is acetylated prior tomethylation. Acetylation may be either N-acetylation or completeacetylation in this case. This is because an O-acetyl group introducedby complete acetylation is immediately detached under strongly basicconditions used for methylation and a methyl group is introduced inplace of the O-acetyl group. N-acetyl groups remain to be attached underthese conditions.

[0077] Those skilled in the art can readily carry out N-acetylation of ahydrazino derivative. For example, N-acetylation of a hydrazinooligosaccharide derivative can be conducted according to the method asdescribed in WO 96/17824 (JP-A 11-501901). Alternatively, a hydrazinooligosaccharide derivative may be completely acetylated by a method wellknown to those skilled in the art, for example, by allowing it to standin a 2:1 mixture of pyridine and acetic acid at 37° C. overnight.

[0078] The present inventors have found another unexpected effect ofacetylation of a nitrogen atom (N) directly bound to a carbon atomderived from a saccharide in a hydrazino derivative. Specifically, thechemical stability of a hydrazino derivative was increased byacetylating a nitrogen atom (N) directly bound to a carbon atom derivedfrom a saccharide in the hydrazino derivative. A phenomenon that anN-acetylated hydrazino derivative is very stable whereas a saccharidehydrazino derivative is unstable in an acidic solution was observed.Furthermore, it is additionally advantageous that an N-acetylatedhydrazino derivative results in a sharper peak or band as compared witha hydrazino derivative that is not N-acetylated. This is because anN-acetylated hydrazino derivative does not have a charge due toprotonation of a nitrogen atom and thus does not interact with a carrierupon chromatography for purification or analysis.

[0079] A monosaccharide at a reducing end of a saccharide having thereducing end can be identified by identifying, in step (e), thecompletely acetylated hydrazino monosaccharide derivative obtained asdescribed above. Furthermore, the position of binding of amonosaccharide at a reducing end to a neighboring monosaccharide can bedetermined by methylating a hydrazino derivative at a hydroxyl group asdescribed above.

[0080] The derivative of the present invention may be purified prior toanalysis. Alternatively, it may not necessarily be purified if a systemin which chromatography is connected to analytical equipment such as gaschromatography/mass spectrometry (GC/MS) or liquid chromatography/massspectrometry (LC/MS) (generically called an on-line mass spectrometricanalysis) is used. The completely acetylated hydrazino monosaccharidederivative may be purified prior to or following acetylation. Thederivative is purified using preferably chromatography, more preferablyhigh performance liquid chromatography.

[0081] A completely acetylated hydrazino monosaccharide derivative canbe used for determining the structure of the monosaccharide. A methodfor determining the structure preferably comprises separation bychromatography and a mass spectrometric analysis. More preferably,separation is accomplished by gas chromatography (GC/MS).

[0082] A derivative is detected using any appropriate technique known tothose skilled in the art. Preferably, the detection is typically carriedout using ultraviolet absorbance at 200 nm or mass spectrometry (MS).Detection of a derivative using an on-line mass spectrometric analysissuch as GC/MS or LC/MS (i.e., directly introducing a compound separatedby chromatography into a mass spectrometer for analysis) is particularlypreferable.

[0083] In a mass spectrometric analysis, a sample in a gas state isionized in vacuo by a method such as electron impact (EI) or chemicalionization (CI), and the resulting ion is detected. In case of anon-line mass spectrometric analysis, a molecule separated bychromatography is ionized by electron impact (EI) or chemical ionization(CI). The amount of a sample required for analysis is usually less than1 pmol. For review on basic equipment, see Cooks, R. G., Glish, G. L.,McLucky, S. A. and Kaiser, R. E., Chemical and Engineering News, Mar.25, 1991, pp. 26-41 (incorporated herein by reference).

[0084] Unlike a general methylation analysis as described in theBackground Art section, it is intended to carry out a methylationanalysis only for a monosaccharide at a reducing end according to themethod for determining a binding position of a saccharide of the presentinvention. The type of a monosaccharide at a reducing end can beidentified by converting an oligosaccharide to a hydrazino derivative,cleaving the resulting hydrazino oligosaccharide derivative by areaction that can cleave a glycosidic linkage, obtaining a hydrazinomonosaccharide derivative, completely acetylating the hydrazinomonosaccharide derivative and subjecting the product to GC/MS (Bendiak,B. and Fang, T. T., Carbohydr. Res. 327, 463-481 (2000)). The positionof binding of a monosaccharide at a reducing end to a neighboringmonosaccharide can be determined by a procedure almost the same as thatof a general methylation analysis. Specifically, analysis is carried outby converting an oligosaccharide to a hydrazino derivative, completelymethylating the resulting hydrazino oligosaccharide derivative, cleavingthe completely methylated hydrazino oligosaccharide derivative by areaction that can cleave a glycosidic linkage, obtaining a partiallymethylated hydrazino monosaccharide derivative, completely acetylatingit and subjecting the partially methylated/acetylated hydrazinomonosaccharide derivative (partially methylated 1-deoxy-1-hydrazinoalditol acetates, or partially methylated 2-deoxy-2-hydrazino alditolacetates, PMHAA) to GC/MS. A saccharide other than the saccharide at thereducing end which may be detected by the above-mentioned procedure as apartially methylated/acetylated saccharide can be distinguished from thepartially methylated/acetylated hydrazino monosaccharide derivativebased on the retention time on GC or the mass spectrum. Distinctionbased on a mass spectrum is particularly effective. For example, amolecular ion mass of PMHAA can be detected by determining a positiveion mass spectrum by chemical ionization using isobutane. If the type ofa monosaccharide at a reducing end has been identified by a reducing endanalysis, the number of possible molecular ion masses for the expectedPMHAAs is limited to 6 at the most. Then, the peak for the PMHAA can bereadily identified by scanning the chromatogram for the mass.

[0085] The position of an acetyl group in a PMHAA can be identifiedbased on a fragmentation pattern obtained by determining an MS/MSspectrum of a detected molecular ion.

[0086] It is basically possible to apply fragmentation patterns forPMAAs upon mass spectrometric analyses and rules thereof, which havebeen conventionally examined in detail, to fragmentation patterns forPMHAAs upon MS/MS. However, there has been no report on obtainment of aPMHAA, and details of the fragmentation patterns unique to the PMHAAsupon mass spectrometric analyses and rules thereof have not beenexamined yet at all.

[0087] A PMHAA can be produced using an N,N′-diacetylated hydrazinooligosaccharide (e.g., 1-deoxy-1-(N,N′-diacetyl hydrazino)-lactitol) asa raw material. The method disclosed in WO 96/17824 (JP-A 11-501901) canbe used as a method for producing an N,N′-diacetylated hydrazinooligosaccharide. An N,N′-diacetylated hydrazino oligosaccharide iscompletely methylated, and a partially methylated hydrazinomonosaccharide is then obtained, for example, by a glycosidiclinkage-cleaving reaction such as methanolysis. A PMHAA can be obtainedby completely acetylating the resulting partially methylated hydrazinomonosaccharide. The thus obtained PMHAA may be used as it is, or it maybe used after further purification by chromatography such as reversephase high performance liquid chromatography.

[0088] One of properties of PMHAAs is that they are less volatile. Dueto this property, mild distillation-removal of a solvent by nitrogenblowing which is required for a PMAA is not necessary. Loss of a sampleis not observed at all even if drying under reduced pressure using acentrifugation concentrator is carried out.

[0089] The present invention also relates to a hydrazine, a kit used inthe method of the present invention as described above. The kit containssaid hydrazine as an essential component, and is for producing ahydrazino monosaccharide derivative or for identifying a monosaccharideat a reducing end of a saccharide and/or for determining a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide. The kit may further contain an additional reagent to beused for a reaction in each step, a reaction vessel, instructions andthe like.

EXAMPLES

[0090] The following examples further illustrate the present inventionin detail but are not to be construed to limit the scope thereof.

Example 1

[0091] Preparation and Analysis of Hydrazino Glucose Derivative

[0092] (a) Preparation of Hydrazone

[0093] 20 μl of a 4 μM 1-pyrenebutanoic acid, hydrazide (MolecularProbes) solution in dimethyl sulfoxide (DMSO) was added to 360 μg ofglucose which had been dried adequately, and glucose was fully dissolvedby sonication. 2 μl of acetic acid was added thereto and the mixture wasstirred adequately. The reaction mixture was then heated at 90° C. for 1hour.

[0094] (b) Reduction of Hydrazone

[0095] The reaction mixture obtained in (a) was dried using acentrifugation concentrator. 20 μl of a solution containing 2.5 Mborane-dimethylamine complex and 30% acetic acid in DMSO was added tothe residue, and the residue was fully dissolved by sonication. Themixture was then heated at 80° C. for 1 hour. The reaction mixture wasdried using a centrifugation concentrator. The residue was re-dissolvedin 50% acetonitrile and purified using normal phase high performanceliquid chromatography. The existence of pyrene-labeled glucose wasconfirmed by determining the molecular weight of the purified productusing a triple-quadrupole ion-spray mass-spectrometer API-300(Perkin-Elmer Sciex) to detect a positive molecular ion (467.0).

[0096] (c) Methanolysis of Pyrene-labeled Glucose

[0097] The purified pyrene-labeled glucose obtained in (b) (10 nmol) wasplaced in a glass test tube and dried. 100 μl of 5% hydrochloricacid-methanol (Nacalai Tesque) was added to the test tube. The tube wassealed and heated at 90° C. for 4 hours. The tube was opened, and thesample was dried using a centrifugation concentrator to obtain acleavage product.

[0098] (d) GC/MS Analysis

[0099] 10 nmol of 1-deoxy-1-(N,N′-diacetyl hydrazino)-¹³C₆-D-glusitol asan internal standard was added to the residue obtained aftermethanolysis of the pyrene-labeled glucose in (c), and the mixture wasdried again. 1-deoxy-1-(N,N′-diacetyl hydrazino)-¹³C₆-D-glusitol wasprepared according to a known method (JP-A 11-501901; incorporatedherein by reference) using ¹³C₆-D-glucose (Aldrich) as a startingmaterial. 100 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the resulting residue. After adequately stirring, the mixturewas incubated at 37° C. for 16 hours. The sample was dried using acentrifugation concentrator. The residue was dissolved by adding 200 μlof chloroform thereto. 1 μl of the solution was analyzed by subjectingit to GC/MS by splitless injection. GC/MS analysis was carried out asfollows:

[0100] System: GCQ (Finnigan MAT)

[0101] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0102] Carrier: Herium (40 cm/sec)

[0103] Ionization: EI

[0104] Injector temperature: 300° C.

[0105] Column initial temperature: 90° C.

[0106] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(4° C./min)-300° C.

[0107] Injection: 1 microliter (splitless injection)

[0108] Main peaks were observed at 16.04 minutes and 21.21 minutes in atotal ion mass chromatogram. A peak was observed at 16.04 minutes in asingle mass chromatogram for a specific ion ([M-42]⁺) at m/z=448 forcompletely acetylated 1-deoxy-1-(N,N′-diacetylhydrazino)-¹²C₆-D-glusitol. This peak was consistent with a peak in asingle mass chromatogram for a specific ion ([M-42]⁺) at m/z=454 forcompletely acetylated 1-deoxy-1-(N,N′-diacetylhydrazino)-¹³C₆-D-glusitol from the internal standard. The peak at 21.21minutes had a main ion of m/z=302 which was consistent with themolecular ion of 1-pyrenebutanoic acid methyl ester.

Example 2

[0109] Hydrazine-immobilized Support

[0110] (a) Preparation of Carbohydrazide-immobilized NovaSyn TG Resin

[0111] 360 mg of NovaSyn TG bromo Resin (Nova Biochem) was suspended ina 0.5 M carbohydrazide (Aldrich) solution in DMSO. The mixture wasshaken at room temperature for 16 hours. The resin was washed insufficient amounts of DMSO, water and ethanol, treated with a 15 mg/mlcesium acetate/dimethylformamide (DMF) solution, and then washed insufficient amounts of DMF, water and ethanol to obtaincarbohydrazide-immobilized NovaSyn TG Resin. The reducing power ofcarbohydrazide being introduced was measured according to thePark-Johnson method (Park, J. T. and Johnson, M. J., J. Biol. Chem.,181, 149-151 (1949)). As a result, a reducing power corresponding to 1.1nmol of 4-methoxyphenylhydrazine hydrochloride was observed for 1 mg ofthe resin.

[0112] (b) Preparation of Hydrazine-immobilized Bio-Rex 70 Resin

[0113] 200 mg of Bio-Rex 70 Resin (Bio-Rad) of which the ion type hadbeen converted into a proton form was suspended in 5 ml ofN,N-dimethylformamide (DMF). 0.5 g of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)(Nacalai Tesque) was added thereto. The mixture was shaken at roomtemperature for 16 hours. The resin was washed in 20 ml of DMF. 5 ml ofa 10% hydrazine (Nacalai Tesque) solution in DMF was then added thereto.The mixture was shaken for additional 5 hours. The resin was washed insufficient amounts of DMF, water, 1 M hydrochloric acid, water andmethanol to obtain hydrazine-immobilized Bio-Rex 70 Resin. The reducingpower of hydrazine being introduced was measured according to thePark-Johnson method (Park, J. T. and Johnson, M. J., J. Biol. Chem.,181, 149-151 (1949)). As a result, a reducing power corresponding to27.6 nmol of 4-methoxyphenylhydrazine hydrochloride was observed for 1mg of the resin.

[0114] (c) Attachment of Saccharide to Resin

[0115] 100 μl of a solution containing 100 mM glucose and 10% aceticacid in DMSO was added to 10 mg of the hydrazine-immobilized Bio-Rex 70Resin obtained in (b). The mixture was heated at 90° C. for 1 hour. Theresin was washed in 50 ml of DMSO. 1 ml of a 1 M sodium boron hydrideaqueous solution was then added the resin. The mixture was allowed tostand at room temperature for 16 hours. The resin was washed insufficient amounts of water, 0.1 M hydrochloric acid, water, andethanol, and dried.

[0116] (d) Acid Hydrolysis of Saccharide-attached Resin

[0117] The dried saccharide-attached resin obtained in (c) wastransferred to a glass test tube. 100 μl of 4 M hydrochloric acid wasadded thereto. The tube was sealed and heated at 100° C. for 4 hours.After the tube was opened, a supernatant was recovered and dried underreduced pressure to obtain a hydrolysis product.

[0118] (e) GC/MS Analysis

[0119] 100 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the residue obtained after hydrolysis in (d). After adequatelystirring, the mixture was allowed to stand at room temperature for 16hours. The sample was dried using a centrifugation concentrator. Theresidue was dissolved by adding 200 μl of acetonitrile thereto. 1 μl ofthe solution was analyzed by subjecting it to GC/MS by splitlessinjection. GC/MS analysis was carried out as follows:

[0120] System: GCQ (Finnigan MAT)

[0121] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0122] Carrier: Herium (40 cm/sec)

[0123] Ionization: EI

[0124] Injector temperature: 300° C.

[0125] Column initial temperature: 90° C.

[0126] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(4° C./min)-300° C.

[0127] Injection: 1 microliter (splitless injection)

[0128] A peak was observed at 14.5 minutes in a single mass chromatogramfor a specific ion ([M-42]⁺) at m/z=448 for completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-D-glusitol. The mass chromatogramfor this peak was consistent with that for completely acetylated1-deoxy1-(N,N′-diacetyl hydrazino)-D-glusitol as a standard.

Example 3

[0129] Methylation Analysis

[0130] (a) Preparation of Pyrene-labeled Lactose

[0131] 10 μl of a solution containing 400 nmol of 1-pyrenebutanoic acid,hydrazide (Molecular Probes) in DMSO was added to 50 nmol of lactosewhich had been dried adequately, and lactose was fully dissolved bysonication. 1 μl of acetic acid was added thereto and the mixture wasstirred adequately. The reaction mixture was then heated at 90° C. for 1hour. After the reaction mixture was dried using a centrifugationconcentrator, 10 μl of a solution containing 2.5 M borane-dimethylaminecomplex and 30% acetic acid in DMSO was added to the residue, and theresidue was fully dissolved by sonication. The mixture was then heatedat 80° C. for 1 hour. The reaction mixture was dried using acentrifugation concentrator. The residue was re-dissolved in 50%acetonitrile and purified using normal phase high performance liquidchromatography. The molecular weight of the purified pyrene-labeledlactose was determined using a triple-quadrupole ion-spraymass-spectrometer API-300. As a result, a positive molecular ion (629.3)was observed.

[0132] (b) Methanolysis of Pyrene-labeled Lactose

[0133] The purified pyrene-labeled lactose obtained in (a) (20 nmol) wasplaced in a glass test tube and dried. 100 μl of 5% hydrochloricacid-methanol (Nacalai Tesque) was added to the test tube. The tube wassealed and heated at 90° C. for 4 hours. The tube was opened, and thesample was dried using a centrifugation concentrator to obtain acleavage product.

[0134] (c) GC/MS Analysis

[0135] 100 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the residue obtained after methanolysis of the pyrene-labeledlactose. After adequately stirring, the mixture was incubated at 37° C.for 16 hours. The sample was dried using a centrifugation concentrator.The residue was dissolved by adding 200 μl of acetonitrile thereto. 1 μlof the solution was analyzed by subjecting it to GC/MS by splitlessinjection. GC/MS analysis was carried out as follows:

[0136] System: GCQ (Finnigan MAT)

[0137] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0138] Carrier: Herium (40 cm/sec)

[0139] Ionization: CI (Isobutane)

[0140] Injector temperature: 300° C.

[0141] Column initial temperature: 90° C.

[0142] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(4° C./min)-300° C.

[0143] Injection: 1 microliter (splitless injection)

[0144] Main peaks were observed at 8.1 minutes, 14.6 minutes and 20.1minutes in a total ion mass chromatogram. A peak was observed at 14.6minutes in a single mass chromatogram for a positive molecular ion([M+H]⁺) at m/z=491 for completely acetylated 1-deoxy-1-(N,N′-diacetylhydrazino)-D-glusitol. Both the retention time and the mass spectrumwere consistent with those for the completely acetylated1-deoxy-1-(N,N′-diacetyl hydrazino)-D-glusitol as a standard.

[0145] (d) Methylation of Pyrene-labeled Lactose

[0146] 100 nmol of the purified pyrene-labeled lactose obtained in (a)was placed in a screw-capped glass test tube and dried under reducedpressure. 200 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the residue. After adequately stirring, the mixture was allowedto stand at room temperature for 16 hours. The sample was dried using acentrifugation concentrator. 100 μl of methanol was added to theresidue, and the mixture was dried again. The thus obtained sample wassubjected to complete methylation treatment of the pyrene-labeledlactose using the method of Anumula et al. (Anumula, K. R. and Taylor,P. B., Anal. Biochem., 203, 101-108 (1992)). A product obtained afterextraction with chloroform was dried under reduced pressure.

[0147] (e) Methanolysis of Methylated Pyrene-labeled Lactose

[0148] The methylated pyrene-labeled lactose obtained in (d)(corresponding to 50 nmol) was placed in a glass test tube and dried.100 μl of 5% hydrochloric acid-methanol (Nacalai Tesque) was added tothe test tube. The tube was sealed and heated at 90° C. for 4 hours. Thetube was opened, and the sample was dried using a centrifugationconcentrator to obtain a cleavage product.

[0149] (f) GC/MS Analysis

[0150] 100 μl of a 2:1 mixture of pyridine and acetic anhydride wasadded to the residue obtained in (e) after methanolysis of themethylated pyrene-labeled lactose. After adequately stirring, themixture was allowed to stand at room temperature for 16 hours. Thesample was dried using a centrifugation concentrator. The residue wasdissolved by adding 200 μl of acetonitrile thereto. 1 μl of the solutionwas analyzed by subjecting it to GC/MS by splitless injection. GC/MSanalysis was carried out as follows:

[0151] System: GCQ (Finnigan MAT)

[0152] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0153] Carrier: Herium (40 cm/sec)

[0154] Ionization: CI(Isobutane)

[0155] Injector temperature: 300° C.

[0156] Column initial temperature: 90° C.

[0157] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(4° C./min)-300° C.

[0158] Injection: 1 microliter (splitless injection)

[0159] An m/z value of 393 corresponding to a positive molecular ionpeak ([M+H]⁺) for4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glusitolwas observed when the main peak observed at 11.3 minutes was subjectedto mass spectrometry.

Example 4

[0160] Preparation of Partially Methylated/acetylated HydrazinoMonosaccharide Derivative (Partially Methylated 1-deoxy-1-hydrazinoAlditol Acetates, PMHAA)

[0161] 10 μmol each of lactose(Galβ1-4Glc) (Nacalai Tesque), mannobiose(Manα1-2Man) (Dextra Laboratories), mannotriose (Manα1-6[Manα1-3]Man)(Dextra Laboratories) and N-acetyl lactosamine (Galβ1-4GlcNAc)(Seikagaku Corporation) was converted to an N,N′-diacetylated hydrazinoderivative according to the method as described in WO 96/17824 (JP-A11-501901). 1 μmol each of N,N′-diacetylated hydrazino derivative of therespective oligosaccharides was completely methylated according to themethod of Anumula et al. (supra). The resulting completely methylatedN,N′-diacetylated hydrazino oligosaccharide derivative was placed in aglass test tube and dried. 500 μl of 5% hydrochloric acid-methanol(Nacalai Tesque) was added to the test tube. The tube was sealed andheated at 90° C. for 4 hours. The tube was opened, and the sample wasdried using a centrifugation concentrator to obtain a cleavage product.300 μl of a 2:1 mixture of pyridine and acetic anhydride was added tothe residue obtained after methanolysis. After adequately stirring, themixture was incubated at 37° C. for 2 hours. The sample was dried usinga centrifugation concentrator. The residue was dissolved by adding 1000μl of an acetonitrile aqueous solution thereto and each PMHAA wasrecovered.

[0162] GC/MS Analysis

[0163] 1 μl of each PMHAA solution in acetonitrile (corresponding to 1nmol of the starting material) was analyzed by subjecting it to GC/MS bysplitless injection. GC/MS analysis was carried out as follows:

[0164] System: GCQ (Finnigan MAT)

[0165] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0166] Carrier: Herium (40 cm/sec)

[0167] Ionization: CI (Isobutane)

[0168] Injector temperature: 300° C.

[0169] Column initial temperature:.90° C.

[0170] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(4° C./min)-300° C.

[0171] Injection: 1 microliter (splitless injection)

[0172] Plural peaks were observed for each PMHAA sample upon GC/MSanalysis. The peaks for PMHAAs were identified based on positivemolecular ions found in the respective chemical ionization mass spectra.

[0173] The results confirmed the following:4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolresulted from lactose;2-O-acetyl-1-deoxy-3,4,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolresulted from mannobiose;3,6-O-diacetyl-1-deoxy-2,4,5-O-trimethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolresulted from mannotriose; and4-O-acetyl-1,2-dideoxy-3,5,6-O-trimethyl-2-(N-methylacetoamido)-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolresulted from N-acetyl lactosamine.

[0174] Upon GC/MS analysis of4-O-acetyl-1-deoxy-2,3,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolobtained from lactose, a positive molecular ion peak ([M+H]⁺) at m/z=393was detected in the chemical ionization mass spectrum for the peak at11.4 minutes. Upon GC/MS analysis of2-O-acetyl-1-deoxy-3,4,5,6-O-tetramethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolobtained from mannobiose, a positive molecular ion peak ([M+H]⁺) atm/z=393 was detected in the chemical ionization mass spectrum for thepeak at 11.1 minutes. Upon GC/MS analysis of3,6-O-diacetyl-1-deoxy-2,4,5-O-trimethyl-1-(N,N′-diacetyl-N′-methylhydrazino)-D-mannitolobtained from mannotriose, a positive molecular ion peak ([M+H]⁺) atm/z=421 was detected in the chemical ionization mass spectrum for thepeak at 13.1 minutes. Upon GC/MS analysis of4-O-acetyl-1,2-dideoxy-3,5,6-O-trimethyl-2-(N-methylacetoamido)-1-(N,N′-diacetyl-N′-methylhydrazino)-D-glucitolobtained from N-acetyl lactosamine, a positive molecular ion peak([M+H]⁺) at m/z=434 was detected in the chemical ionization massspectrum for the peak at 14.5 minutes.

Example 5

[0175] Preparation and Methylation Analysis of UltravioletAbsorbance-labeled Hydrazide Derivative

[0176] 25 μl of a solution containing 25 μmol of benzoylhydrazine (TokyoKasei Kogyo) in dimethyl sulfoxide (DMSO) was added to 5 μmol of glucose(Nacalai Tesque), sophorose (Glcβ1-2Glc, Sigma), laminaribiose(Glcβ1-3Glc, Seikagaku Corporation), maltose (Glcα1-4Glc, Kanto Kagaku)or isomaltose (Glcα1-6Glc, Seikagaku Corporation) which had been driedadequately, and the saccharide was fully dissolved by sonication. 2.5 μlof acetic acid was added thereto and the mixture was stirred adequately.The reaction mixture was then heated at 90° C. for 1 hour. The reactionmixture was dried using a centrifugation concentrator. 50 μl of asolution containing 2.5 M borane-dimethylamine complex and 30% aceticacid in DMSO was added to the residue, and the residue was fullydissolved by sonication. The mixture was then incubated at 37° C. for 16hours. The reaction mixture was dried using a centrifugationconcentrator. Addition of acetonitrile and drying were repeated. Theresidue was dissolved in 100 μl of water. Excess reagents were removedby three rounds of extraction with 300 μl of water-saturated ethylacetate. The method as described in WO 96/17824 (JP-A 11-501901) wasused for carrying out N-acetylation. After desalting using Dowex 50W-X8(Muromachi Kagaku) followed by further purification using HPLC,N-acetylated benzoylhydrazine saccharide derivatives were obtained. HPLCwas carried out as follows:

[0177] Pump: LC6A (Shimadzu)

[0178] Column: Asahipak NH2P-50 (4.6 mm i.d.×250 mm) (Showa Denko)

[0179] Solvent A: Acetonitrile/water, 95:5

[0180] Solvent B: Acetonitrile/water, 1:1

[0181] Flow rate: 1 ml/min

[0182] Temperature: 40° C.

[0183] Gradient: 0-100% Solvent B in 30 min

[0184] Detection: Absorbance at 270 nm

[0185] 1 μmol each of the N-acetylated benzoylhydrazine saccharidederivatives was completely methylated according to the method of Anumulaet al. (supra). A 1/20 amount of the resulting completely methylatedN-acetylated benzoylhydrazine saccharide derivative was placed in aglass test tube and dried. 100 μl of a 80% acetic acid aqueous solutioncontaining 0.5 M hydrochloric acid was added to the test tube. The tubewas sealed and heated at 100° C. for 6 hours. The tube was opened, andthe sample was dried using a centrifugation concentrator to obtain acleavage product. 200 μl of a 2:1 mixture of pyridine and aceticanhydride was added to the residue. After adequately stirring, themixture was incubated at 37° C. for 16 hours. The sample was dried usinga centrifugation concentrator. The residue was dissolved by adding 100μl of an acetonitrile aqueous solution thereto to recover PMHAA.

[0186] GC/MS Analysis

[0187] 1 μl of the PMHAA solution in acetonitrile (corresponding to 1nmol of the starting material) was analyzed by subjecting it to GC/MS bysplitless injection. GC/MS analysis was carried out as follows:

[0188] System: GCQ (Finnigan MAT)

[0189] Column: DB-5 (5% diphenyl-95% dimethyl polysiloxane, 0.25 mmi.d.×30 m, 0.25 micrometer film thickness) (J & W Scientific)

[0190] Carrier: Herium (40 cm/sec)

[0191] Ionization: CI (Isobutane)

[0192] Injector temperature: 300° C.

[0193] Column initial temperature: 90° C.

[0194] Time program: 90° C. for 2 min, 90° C.-(24° C./min)-210° C., and210° C.-(12° C./min)-300° C.

[0195] Injection: 1 microliter (splitless injection)

[0196] Plural peaks were observed in a total mass chromatogram for eachPMHAA. A single peak was observed at 9.2 minutes in a single masschromatogram for a positive molecular ion ([M+H]⁺) at m/z=393 for theexpected PMHAA for the sample from N-acetylated benzoylhydrazineglucose. Single peaks were observed at 9.4 minutes, 9.5 minutes, 9.6minutes and 10.2 minutes in single mass chromatograms for positivemolecular ions ([M+H]⁺) at m/z=393 for the expected PMHAAs for theremaining four samples from N-acetylated benzoylhydrazine sophorose,N-acetylated benzoylhydrazine laminaribiose, N-acetylatedbenzoylhydrazine maltose and N-acetylated benzoylhydrazine isomaltose,respectively. The mass spectra for PMHAA peaks for respective sampleswere clearly different each other among the samples. Accordingly, it wasdemonstrated that the position of an acetyl group can be identifiedbased on the difference in a mass spectrum or a fragmentation pattern ofPMHAA.

Example 6

[0197] Preparation and Stability Test of N-acetylated Labeled HydrazinoDerivative

[0198] 100 μl of a solution containing 100 μmol of benzoylhydrazine(Tokyo Kasei Kogyo) in DMSO was added to 50 μmol of N-acetyl lactosamine(Seikagaku Corporation, hereinafter referred to as LN) which had beendried adequately, and the saccharide was fully dissolved by sonication.80 μl of DMSO and 20 μl of acetic acid were added thereto and themixture was stirred adequately. The reaction mixture was then heated at90° C. for 1 hour. The reaction mixture was dried using a centrifugationconcentrator while heating. 500 μl of a 25% acetonitrile aqueoussolution containing 2 M sodium boron hydride (Nacalai Tesque) was addedto the residue, and the mixture was adequately stirred and allowed tostand at room temperature overnight. 500 μl of pure water was added tothe reaction mixture. The reaction mixture was then neutralized byadding acetic acid dropwise to make the pH of the solution to about 5.800 μl of the neutralized solution was placed in a test tube. 1 ml ofpure water was further added thereto for dilution. The method asdescribed in WO 96/17824 (JP-A 11-501901) was used for carrying outN-acetylation. After desalting using Dowex 50W-X8 (Muromachi Kagaku)followed by further purification using HPLC, an N-acetylation product ofa benzoylhydrazine derivative of LN (hereinafter referred to as LNbenzoylhydrazine derivative) was obtained. On the other hand, an LNbenzoylhydrazine derivative was prepared without N-acetylation. 100 μlof a solution containing 100 μmol of benzoylhydrazine (Tokyo KaseiKogyo) in DMSO was added to 50 μmol of LN (Seikagaku Corporation) whichhad been dried adequately, and the saccharide was fully dissolved bysonication. 80 μl of DMSO and 20 μl of acetic acid were added theretoand the mixture was stirred adequately. The reaction mixture was thenheated at 90° C. for 1 hour. The reaction mixture was dried using acentrifugation concentrator while heating. 500 μl of a 25% acetonitrileaqueous solution containing 2 M sodium boron hydride was added to theresidue, and the mixture was adequately stirred and allowed to stand atroom temperature overnight. 500 μl of pure water was added to thereaction mixture. The reaction mixture was then neutralized by addingacetic acid dropwise to make the pH of the solution to about 5. 200 μlof the neutralized reaction mixture was dried using a centrifugationconcentrator and purified using HPLC to obtain an LN benzoylhydrazinederivative. HPLC was carried out as follows:

[0199] Pump: LC6A (Shimadzu)

[0200] Column: Asahipak NH2P-50 (4.6 mm i.d.×250 mm) (Showa Denko)

[0201] Solvent A: Acetonitrile/water, 95:5

[0202] Solvent B: Acetonitrile/water, 1:1

[0203] Flow rate: 1 ml/min

[0204] Temperature: 40° C.

[0205] Gradient: 0-100% Solvent B in 30 min

[0206] Detection: Absorbance at 270 nm

[0207] 25 nmol each of the purified LN benzoylhydrazine derivative andthe N-acetylation product thereof was dissolved in 5 μl of a 10, 30 or50% acetic acid solution in DMSO. The solution was incubated at 37° C.overnight. The solution was dried and re-dissolved in 5 μl of purewater. 0.5 μl of the solution was spotted onto HPTLC (Merck) anddeveloped using a 80% acetonitrile aqueous solution. Neutral sugars weredetected according to the orcinol-sulfuric acid method.

[0208] Results of thin-layer chromatography are shown in FIG. 1. Thefollowing samples were developed in the lanes in FIG. 1: lane 1: theuntreated LN benzoylhydrazine derivative; lane 2: the LNbenzoylhydrazine derivative incubated in a solution containing 10%acetic acid in DMSO; lane 3: the LN benzoylhydrazine derivativeincubated in a solution containing 30% acetic acid in DMSO; lane 4: theLN benzoylhydrazine derivative incubated in a solution containing 50%acetic acid in DMSO; lane 5: the untreated N-acetylated LNbenzoylhydrazine derivative; lane 6: the N-acetylated LNbenzoylhydrazine derivative incubated in a solution containing 10%acetic acid in DMSO; lane 7: the N-acetylated LN benzoylhydrazinederivative incubated in a solution containing 30% acetic acid in DMSO;and lane 8: the N-acetylated LN benzoylhydrazine derivative incubated ina solution containing 50% acetic acid in DMSO.

[0209] The LN benzoylhydrazine derivative was converted under acidicconditions into a substance that exhibited lower mobility on TLC,whereas no change was observed for the N-acetylated LN benzoylhydrazinederivative. Thus, the chemical stability of the N-acetylatedbenzoylhydrazine derivative was demonstrated.

[0210] Industrial Applicability

[0211] The present invention provides a means that enables determinationof the type and the substitution position of a monosaccharide at areducing end of a saccharide even if it is not isolated.

1. A method for producing a hydrazino monosaccharide derivative, themethod comprising at least: (a) reacting a saccharide having a reducingend with a hydrazine of formula (I) to produce a hydrazone: NH₂-NR¹(R²)  (I) wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a reaction that can cleave aglycosidic linkage; and R² is hydrogen or an alkyl group containing 1-8carbon atoms; (b) reducing the hydrazone obtained in step (a) to ahydrazino derivative; and (c) cleaving the hydrazino derivative obtainedin step (b) by the reaction that can cleave a glycosidic linkage toobtain a hydrazino monosaccharide derivative.
 2. The method according toclaim 1, which comprises N-acetylating the hydrazino derivative obtainedin step (b) before subjecting it to step (c).
 3. The method according toclaim 1, which comprises methylating a hydroxyl group of the hydrazinoderivative obtained in step (b) or an N-acetylation product of thehydrazino derivative before subjecting it to step (c).
 4. The methodaccording to claim 1, wherein R¹ is an acyl group.
 5. The methodaccording to claim 1, wherein R¹ is an acyl group that has a ultravioletor visible-absorbing substance, a fluorescent dye or an immobilizationsupport as its portion.
 6. A method for identifying a monosaccharide ata reducing end of a saccharide having the reducing end and/or fordetermining a position of binding of a monosaccharide at a reducing endto a neighboring monosaccharide, the method comprising at least: (a)reacting a saccharide having a reducing end with a hydrazine of formula(I) to produce a hydrazone: NH₂—NR¹(R²)   (I) wherein R¹ is a groupother than hydrogen that has a detectable label and/or an immobilizationsupport as its portion or can bind to a detectable label and/or animmobilization support; the bond between R¹ and N is a bond that iscleavable by a reaction that can cleave a glycosidic linkage; and R² ishydrogen or an alkyl group containing 1-8 carbon atoms; (b) reducing thehydrazone obtained in step (a) to a hydrazino derivative; (c) cleavingthe hydrazino derivative obtained in step (b) by the reaction that cancleave a glycosidic linkage to obtain a hydrazino monosaccharidederivative; (d) completely acetylating the hydrazino monosaccharidederivative obtained in step (c); and (e) identifying the completelyacetylated hydrazino monosaccharide derivative obtained in step (d). 7.The method according to claim 6, which comprises N-acetylating thehydrazino derivative obtained in step (b) before subjecting it to step(c).
 8. The method according to claim 6, which comprises methylating ahydroxyl group of the hydrazino derivative obtained in step (b) or anN-acetylation product of the hydrazino derivative before subjecting itto step (c), wherein a position of binding of a monosaccharide at areducing end to a neighboring monosaccharide is determined in step (e).9. The method according to claim 6, wherein the identification in step(e) is carried out using gas chromatography/mass spectrometry (GC/MS).10. A method for labeling a saccharide having a reducing end, the methodcomprising at least: (a) reacting a saccharide having a reducing endwith a hydrazine of formula (I) to produce a hydrazone: NH₂—NR¹(R²)  (I) wherein R¹ is a group other than hydrogen that has a detectablelabel and/or an immobilization support as its portion or can bind to adetectable label and/or an immobilization support; the bond between R¹and N is a bond that is cleavable by a chemical reaction that can cleavea glycosidic linkage; and R² is hydrogen or an alkyl group of 1-8 carbonatom(s); (b) reducing the hydrazone obtained in step (a) to a hydrazinoderivative; and (c) N-acetylating the hydrazino derivative obtained instep (b).
 11. A hydrazine of formula (I) used in the method defined byclaim 1: NH₂—NR¹(R²)   (I) wherein R¹ is a group other than hydrogenthat has a detectable label and/or an immobilization support as itsportion or can bind to a detectable label and/or an immobilizationsupport; the bond between R¹ and N is a bond that is cleavable by areaction that can cleave a glycosidic linkage; and R² is hydrogen or analkyl group containing 1-8 carbon atoms.
 12. A kit for producing ahydrazino monosaccharide derivative, which contains the hydrazinedefined by claim
 11. 13. A kit for identifying a monosaccharide at areducing end of a saccharide and/or for determining a position ofbinding of a monosaccharide at a reducing end to a neighboringmonosaccharide, which contains the hydrazine defined by claim
 11. 14. Akit for labeling a saccharide having a reducing end, which contains thehydrazine defined by claim
 11. 15. A saccharide of formula (III):R⁴—N(Ac)—NHR¹   (III) wherein Ac is an acetyl group; R¹ is a group otherthan hydrogen that has a detectable label and/or an immobilizationsupport as its portion or can bind to a detectable label and/or animmobilization support; the bond between R¹ and N is a bond that iscleavable by a chemical reaction that can cleave a glycosidic linkage;R⁴ is a group that may have a glycosidic linkage with a saccharide andfrom which one hydrogen atom linked to the C-1 position of a 1-deoxyaldose or one hydrogen atom linked to the C-2 carbon of a 2-deoxy ketoseis removed, excluding a case where R¹ is an acetyl group, and R⁴ is agroup that does not have a glycosidic linkage with a saccharide and fromwhich one hydrogen atom linked to the C-1 position of a 1deoxy aldose orone hydrogen atom linked to the C-2 carbon of a 2-deoxy ketose isremoved.
 16. The saccharide according to claim 15, wherein R¹ is an acylgroup.